A Fluorescent Immunochromatography Kit for Detection of Canine Parvovirus / Canine Coronavirus

Abstract

The present invention discloses a fluorescence immunochromatography detection kit for canine parvovirus / canine coronavirus, belonging to the technical field of animal disease detection. The kit utilizes fluorescence immunochromatography technology. By coating specific antibodies against canine parvovirus and canine coronavirus on a nitrocellulose membrane and combining with a fluorescence label, rapid and accurate detection of the two viruses in canine fecal samples is achieved. Its innovation lies in optimizing the antibody combination and reaction system, significantly improving the detection sensitivity and specificity, and effectively avoiding cross-reaction. The kit is easy to operate, does not require professional equipment, can provide test results within 15 - 20 minutes, is suitable for on-site rapid screening in places such as pet hospitals, animal disease prevention and control institutions, and farms, provides a powerful tool for the early diagnosis and prevention and control of canine parvovirus and canine coronavirus, and has good application prospects and popularization value.

Description

Technical Field

[0001] This application relates to the technical field of animal disease detection, and specifically relates to a fluorescence immunochromatography canine parvovirus / canine coronavirus detection kit. Background Art

[0002] Canine parvovirus (CPV) belongs to the genus Parvovirus in the family Parvoviridae and is a single-stranded deoxyribonucleic acid virus. It is highly contagious to dogs and mainly causes diseases such as hemorrhagic enteritis and acute myocarditis in dogs. Canine coronavirus (CCV) belongs to the genus Coronavirus in the family Coronaviridae and mainly causes varying degrees of gastroenteritis in dogs, characterized by fatal watery diarrhea clinically. Sick dogs will suddenly fall ill, showing listlessness, loss of appetite, vomiting, and discharging foul-smelling, soft and mucus-containing feces. In severe cases, it will also lead to dehydration and weight loss.

[0003] Traditional clinical symptom observation is difficult to accurately distinguish between canine parvovirus and canine coronavirus infections because the symptoms of the two are somewhat similar and may be confused with other canine diseases, easily leading to misdiagnosis. Using the method of virus isolation and identification requires professional laboratory equipment and technical personnel, with complex operations and a long time-consuming process. Usually, it takes several days or even longer to obtain results, making it difficult to meet the needs of rapid diagnosis. Using serological tests such as neutralization tests and complement fixation tests, although they have high specificity, they also have problems such as cumbersome operations, long detection cycles, and high requirements for laboratory conditions, and are not convenient for widespread application in grass-roots units and pet hospitals. Using molecular biology detection such as polymerase chain reaction (PCR) technology, although it has high sensitivity and specificity, it requires professional molecular biology laboratory equipment and professional technical personnel for operation, with high detection costs, and strict requirements for sample collection, transportation, and storage conditions, restricting its rapid application in the clinical field.

[0004] Fluorescence immunochromatography is an immunological detection technology based on the specific binding of antigens and antibodies. Its basic principle is to use a fluorescent label to label an antibody or antigen. When the target antigen or antibody in the sample binds to the fluorescent-labeled specific antibody or antigen, under the action of chromatography, a detectable fluorescent signal is formed. This method usually consists of a sample pad, a conjugate pad, a reaction pad, an absorbent pad, etc. The sample reacts with the reagents on each pad during the chromatography process, and finally the result is judged by the presence or absence or intensity of the fluorescent signal.

[0005] Combined with the relationship between the intensity of the fluorescent signal and the concentration of the target substance, fluorescence immunochromatography can also achieve quantitative detection of virus antigens or antibodies in the sample, providing a more comprehensive reference basis for the diagnosis and treatment of diseases. The development and application prospects of fluorescence immunochromatography. Summary of the Invention

[0006] In view of this, the present invention provides a fluorescence immunochromatography canine parvovirus / canine coronavirus detection kit. This kit utilizes fluorescence immunochromatography technology. By coating specific antibodies against canine parvovirus and canine coronavirus on a nitrocellulose membrane and combining with a fluorescence label, it realizes the rapid and accurate detection of the two viruses in canine fecal samples. Its innovation lies in optimizing the antibody combination and reaction system, significantly improving the detection sensitivity and specificity, and effectively avoiding cross-reactions. The kit is easy to operate, does not require professional equipment, can provide a detection result within 15 - 20 minutes, is suitable for on-site rapid screening in places such as pet hospitals, animal disease prevention and control institutions, and farms, provides a powerful tool for the early diagnosis and prevention and control of canine parvovirus and canine coronavirus, and has good application prospects and promotion value.

[0007] One object of the present invention is to provide a fluorescence immunochromatography canine parvovirus / canine coronavirus detection kit, including a VP2 antibody labeled with fluorescent microspheres, an M antibody labeled with fluorescent microspheres, a goat anti-mouse IgG antibody, a nitrocellulose membrane, a sample pad, a conjugate pad, an absorbent pad, and a PVC backing. The VP2 antibody labeled with fluorescent microspheres is an antibody obtained by conjugating VP2-VH shown in SEQ ID NO:1 and VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys. The M antibody labeled with fluorescent microspheres is an antibody obtained by conjugating M-VH shown in SEQ ID NO:3 and M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys.

[0008] One object of the present invention is to provide an assembly method for a fluorescence immunochromatography canine parvovirus / canine coronavirus detection test strip, including: using a dotting machine to spray a buffer solution of 1 mg / mL VP2-VH and VP2-VL on the test line (T1 line) of the NC membrane, a buffer solution of 1 mg / mL M-VH and M-VL on the test line (T2 line) of the NC membrane, and goat anti-mouse IgG on the quality control line (C line), with a spraying amount of 0.4 μL / cm. After spraying, the NC membrane is dried at 37°C for 2 h. The distance between the T1 and T2 lines is 6 mm, and the distance between the T2 line and the C line is also 6 mm.

[0009] The VP2 antibody labeled with fluorescent microspheres and the M antibody labeled with fluorescent microspheres are mixed in equal mass and then sprayed on the conjugate pad with a dotting machine. The spraying concentration is usually 1 mg / mL, and the spraying amount is 0.4 μL / cm. It is dried at 37°C for 2 hours. The sample pad and the absorbent pad are respectively pasted on the PVC backing with the nitrocellulose membrane, and cut into strips 4 mm wide with a strip cutter, thus making the test strip.

[0010] Among them, the VP2 antibody labeled with fluorescent microspheres is an antibody obtained by conjugating VP2-VH shown in SEQ ID NO:1 and VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys. The M antibody labeled with fluorescent microspheres is an antibody obtained by conjugating M-VH shown in SEQ ID NO:3 and M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys.

[0011] In a specific embodiment, the VP2-VH shown in SEQ ID NO:1 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:5 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VH, transferring pET-28a-VP2-VH into Escherichia coli, screening positive clones, culturing and inducing the expression of the positive clones, collecting the bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VH from the lysate.

[0012] In a specific embodiment, the VP2-VL shown in SEQ ID NO:2 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:6 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VL, transferring pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing the expression of the positive clones, collecting the bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VL from the lysate.

[0013] In a specific embodiment, the VP2-VH shown in SEQ ID NO:3 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:7 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VH, transferring pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing the expression of the positive clones, collecting the bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VH from the lysate.

[0014] In a specific embodiment, the M-VL shown in SEQ ID NO:4 provided by the present invention is obtained by cloning the target sequence shown in SEQ ID NO:8 into pET-28a(+) to obtain the expression plasmid pET-28a-M-VL, transferring pET-28a-M-VL into Escherichia coli, screening positive clones, culturing and inducing the expression of the positive clones, collecting the bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the M-VL from the lysate.

[0015] In a specific embodiment, the VP2 antibody labeled with fluorescent microspheres provided by the present invention is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azide peptide to obtain microspheres conjugated with cyclopentapeptide; then reacting the activation solution of the VP2-VH with the microspheres conjugated with cyclopentapeptide, and then adding the activation solution of the VP2-VL to react.

[0016] In a specific embodiment, the M antibody labeled with fluorescent microspheres provided by the present invention is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azide peptide to obtain microspheres conjugated with cyclopentapeptide; then reacting the activation solution of the M-VH with the microspheres conjugated with cyclopentapeptide, and then adding the activation solution of the M-VL to react.

[0017] One of the purposes of the present invention is to provide the use of antibodies against canine parvovirus and / or antibodies against canine coronavirus in the preparation of a fluorescent immunochromatographic test kit for detecting canine parvovirus / canine coronavirus. The antibodies against canine parvovirus include VP2-VH shown in SEQ ID NO:1 and VP2-VL shown in SEQ ID NO:2, and the antibodies against canine coronavirus include M-VH shown in SEQ ID NO:3 and M-VL shown in SEQ ID NO:4. Beneficial effects

[0018] The antibodies against canine parvovirus and antibodies against canine coronavirus provided by the present invention are designed respectively against VP2 and M proteins, and unconventional VH peptides and VL peptides are obtained by recombinant expression. These variable regions not only have high specificity, but also can act as raw materials for quickly capturing and recognizing viruses to detect these two viruses.

[0019] The magnetic fluorescent microsphere-labeled antibody provided by the present invention, compared with the antibodies provided by the prior art and the prepared magnetic fluorescent microsphere-labeled antibody, can not only detect canine parvovirus / canine coronavirus simultaneously, but also exclude interference and has higher sensitivity. Moreover, the fluorescent signal of the magnetic fluorescent microspheres is stronger than that of traditional colloidal gold or enzyme labeling, and the background noise is lower.

[0020] In addition, the present invention involves two detection lines (T1 and T2) through VP2-VH, VP2-VL, M-VH, M-VL, the VP2 antibody labeled with fluorescent microspheres and the M antibody labeled with fluorescent microspheres, which are respectively used to detect canine parvovirus and canine coronavirus, realizing simultaneous detection. Compared with the traditional single-virus test strip, this dual detection card can detect two viruses simultaneously, reducing the detection time and cost.

[0021] Moreover, the test strip provided by the present invention adopts a highly specific antibody-antigen reaction, reducing non-specific interference. Description of the Drawings

[0022] Figure 1 SDS-PAGE diagrams of recombinantly expressed VP2-VH, VP2-VL, M-VH, and M-VL.

[0023] Figure 2 WB diagrams of recombinantly expressed VP2-VH, VP2-VL, M-VH, and M-VL.

[0024] Figure 3 Infrared diagrams of alkynyl fluorescent microspheres (upper curve) and microspheres conjugated with cyclopentapeptide (lower curve).

[0025] Figure 4 SDS-PAGE diagrams of VP2 antibody labeled with fluorescent microspheres and M antibody labeled with fluorescent microspheres.

[0026] Figure 5 Standard curves for detecting VP2 and M proteins using test strips provided for the experimental group and the control group, respectively.

[0027] Figure 6 Physical diagrams of test strips provided for the experimental group and the control group for recombinantly expressed porcine parvovirus VP2 protein, COVID-19 N protein, canine parvovirus VP2 protein, and canine coronavirus M protein, respectively. Detailed Description of the Invention

[0028] In order to make the objectives, technical solutions, and advantages of this application clearer, the following further details this application in combination with examples. It should be understood that the specific examples described herein are only used to explain this application and are not used to limit this application. Reagents not described in detail and individually in this application are all conventional reagents and can be obtained from commercial sources; methods not described in detail and specifically are all conventional experimental methods and can be learned from the prior art.

[0029] Example 1: Screening of Specific Antibodies against Canine Parvovirus and Canine Coronavirus

[0030] The present invention screened and prepared antibodies against Canine parvovirus Capsid protein VP2 (GenBank: QDA34010.1) and antibodies against Canine coronavirus Membrane protein M (GenBank: BAA02413.1).

[0031] Through the analysis of VP2 antigen epitopes and M antigen epitopes, VH (heavy chain variable region) and VL (light chain variable region) that can specifically bind to the antigen were screened using a phage display library or a synthetic antibody library.

[0032] The VH and VL sequences screened against the VP2 antigen are as follows:

[0033] VP2-VH: QSELSSSGAEDASPGASDSMSCKASGDFDSRYTEMHWDKQRPGQGLEWIGFITPSRGYTDYDQRFDRDATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYEDYYSDEYWGQGTTLTVSS, SEQ ID NO:1

[0034] VP2-VL: ESDLTQSPEDLSLSPEEDATLSCDASQDVDEYDSWYQQKPGQAPRLLIYDESNDATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQESNDWPSLTFGAGTKLELK, SEQ ID NO:2

[0035] The VH and VL sequences against M antigen obtained by screening are as follows:

[0036] M-VH:

[0037] ESDLVESGGGLVQPGGSLRLSCAASGDFDESYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYGDYWGQGTLVTVSS, SEQ ID NO:3

[0038] M-VL:

[0039] DSDMTQSPEDLSLSPEEDATLSCDASQDVDEYDSWYQQKPGKAPKLLIYDESNDATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQESNDWPSLTFGAGTKLELK, SEQ ID NO:4

[0040] VH and VL are linked by a linker peptide (such as (G4S)3, i.e., GGGGSGGGGSGGGGS) to form a single-chain antibody (scFv) of VH-Linker-VL. The scFv can be prepared by expression in Escherichia coli, yeast or mammalian cells, or the VH, VL and linker peptide chains can be obtained by recombinant expression of proteins, and the corresponding single-chain antibody can be obtained by condensation reaction.

[0041] Example 2. Preparation of VP2-VH, VP2-VL, M-VH, M-VL

[0042] 1. Target sequence

[0043] The purpose of codon optimization is to improve the expression efficiency of the target protein in Escherichia coli. The following is the nucleotide sequence after codon optimization (suitable for the Escherichia coli expression system):

[0044] Target sequence of VP2-VH:

[0045] cagtcggaactgtcgtcgagcggtgccgaagacgcgtcgccgggcgcgtcggacagcatgagctgcaaagcgagcggtgacttcgactcgcgtacggaacgcatgcacgactggcagcaacgtccgggccagggccagggcctggagtggatcggcgacatcaccccgtcgcgctacaccgactacgaccaacggttcgaccgagacgcgacgctgacgaccgacaagtcgtcgtcgaccgcttacatgcagctgtcgtcgctggactcggaagactcggcggtgtactgctgcgcgcgttactacgaggactactactcggacgagtactggggccaggggacgacgctggtgacgtcgtcg, SEQ IDNO:5

[0046] Target sequence of VP2-VL

[0047] gaaagcgacctgacccagtcgccggaagacctgtcgctgtcgccggaagacgcgacgcgctgcgacgcgtcgcaggacgtcgacgagtacgactcgtggtaccagcagaaaccgggccaggccccgcgcctgctgatctacgacgagtccaacgacgcgacgggcatcccggcccgcttcagcggctcgggcagcgggaccgacttcacgctgacgatctcgtccctggaaccggacgacgcggtgtactgctgcagcaagagtccaacgactggccgtcgctgacgttcggcgcggggaccaagctggaactgaa, SEQ ID NO:6

[0048] Target sequence of M-VH

[0049] gaaagcgacctggtggaatcgggcggtggtctggtgcagccgggcggttcgctgcgctcgagctgcgcggcgagcggtgacttcgacgaatcgtacgccatgtcttgggtgcggcaggccccgggcaaaggcctggagtgggtgtcggccatctcgggcagcggtggttcgacgtacgccgactcggtgaagggccgcttcacgatctcgcgcgacaactcgaagaacacgctgtacctgcagatgaactcgctgcgcgccgaggacaccgccgtgtactgctgcgcgcgcgactacggcgactactggggccaggggacgacgctggtgacggtgtcgtcg, SEQ ID NO:7

[0050] Target sequence of M-VL

[0051] gactcggacatgacccagtcgccggaagacctgtcgctgtcgccggaagacgcgacgcgctgcgacgcgtcgcaggacgtcgacgagtacgactcgtggtaccagcagaaaccgggccaggccccgcgcctgctgatctacgacgagtccaacgacgcgacgggcatcccggcccgcttcagcggctcgggcagcgggaccgacttcacgctgacgatctcgtccctggaaccggacgacgcggtgtactgctgcagcaagagtccaacgactggccgtcgctgacgttcggcgcggggaccaagctggaactgaa, SEQ ID NO:8

[0052] Amplify the target fragment

[0053] Perform PCR amplification using the primers shown in Table 1, verify the digestion effect by agarose gel electrophoresis, and recover the target sequences of VP2-VH with restriction sites, the target sequences of VP2-VL with restriction sites, the target sequences of M-VH with restriction sites, and the target sequences of M-VL with restriction sites. Among them, the PCR amplification system is 2.0 μL of 10×PCR Buffer, 1.6 μL of (25 mM) MgCl2, 0.4 μL of dNTP Mix (10 mM each of the four deoxynucleoside triphosphates), 0.4 μL of upstream primer (10 μM), 0.4 μL of downstream primer 2 (10 μM), 0.1 μL of Taq DNA polymerase (5 U / μL), 50 ng of template DNA, and ddH2O is added to make up to 20 μL. The PCR amplification program is initial denaturation at 95 °C for 5 minutes; denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 1 minute to form 1 cycle. After a total of 35 cycles, final extension is carried out at 72 °C for 5 minutes, and then stored at 4 °C. The capital letters in Table 1 are restriction sites.

[0054] Table 1

[0055] 3. Digest pET-28a(+)

[0056] Perform double digestion of pET-28a(+) (JHBIO, JH1207) with NdeI and XhoI restriction endonucleases. The digestion reaction system is: 1 μg of pET-28a(+), 1 μL of (10 U / μL) NdeI, 1 μL of (10 U / μL) XhoI, 5 μL of 10× buffer, and deionized water is added to make up to 50 μL. Digest at 37 °C for 2 h. After digestion, verify the digestion effect by agarose gel electrophoresis and recover the pET-28a(+) fragment.

[0057] 4. Recombinant expression steps

[0058] Mix the target sequences of VP2-VH with restriction sites, the target sequences of VP2-VL with restriction sites, the target sequences of M-VH with restriction sites, and the target sequences of M-VL with restriction sites with the pET-28a(+) fragment at a molar ratio of 3:1, and use T4 DNA ligase to carry out a ligation reaction at 16 °C for 4 hours or overnight. The reaction system is 50 ng of pET-28a(+) fragment, 150 ng of the inserted fragment, 1 μL of (400 U / μL) T4 DNA ligase, 2 μL of 10× ligation buffer, and deionized water is added to make up to 20 μL.

[0059] Transform the ligation product into competent Escherichia coli BL21(DE3), screen for positive clones on an LB plate containing kanamycin (50 μg / mL), and verify the positive clones by colony PCR or sequencing. Pick a positive clone and inoculate it into an LB liquid medium containing kanamycin (50 μg / mL), and culture it overnight with shaking at 37°C. Inoculate the overnight culture into fresh LB medium at a ratio of 1:100, and culture it with shaking at 37°C until the OD600 reaches 0.6 - 0.8. Add IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.5 mM to induce protein expression, reduce the culture temperature to 16 - 25°C, and continue to culture with shaking for 12 - 16 hours to reduce inclusion body formation. Centrifuge the culture at 4°C and 5000 g for 10 min to collect the cells. Wash the cells with pre-cooled PBS buffer and centrifuge again to collect the cells.

[0060] Resuspend the cells in lysis buffer (such as 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). Use an ultrasonic disruptor to lyse the cells (operate on ice, power 200 W, work for 2 s, interval 5 s, total time 10 min). Centrifuge (4°C, 12000 g, 30 min) to collect the supernatant. Load the supernatant onto a pre-equilibrated Ni-NTA affinity chromatography column. Wash the column with wash buffer (such as 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0) to remove non-specifically bound proteins. Elute the target protein with elution buffer (such as 50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, pH 8.0). Dialyze the eluted protein into storage Tris-HCl buffer to remove imidazole. Concentrate the protein using an ultrafiltration centrifugal tube and freeze-dry it to obtain the target protein.

[0061] Prepare a protein solution with Tris-HCl buffer at 2 mg / mL for SDS-PAGE, and the results are as Figure 1 shown. The control group is the expression result of Escherichia coli transformed with pET-28a(+), and the other lanes are the results of VP2-VH, VP2-VL, M-VH, and M-VL respectively. It can be seen that protein bands of 16,500 Da, 14,300 Da, 15,400 Da, and 14,300 Da appear respectively, which are consistent with the theoretical sizes.

[0062] The immunoaffinity of VP2-VH and VP2-VL with VP2 protein (CSB-YP355948JAK, CUSABIO), and the immunoaffinity of M-VH and M-VL with M protein (CSB-CF752619CHAH, CUSABIO) were verified by WB method. VP2-VH, VP2-VL, M-VH and M-VL were separated by 12% SDS-PAGE and transferred to PVDF membrane respectively; blocked with PBS buffer containing 3% BSA for 2h; then incubated PVDF membrane with VP2 protein or M protein at 4℃ overnight; washed the membrane 5 times with PBST, and incubated with HRP-labeled anti-His antibody at 37℃ for 1h; washed the membrane 5 times again, used ECL substrate colorimetric solution, and exposed to light for color in a dark box. Figure 2 As shown, VP2-VH and VP2-VL have immunoaffinity with VP2, and M-VH and M-VL have immunoaffinity with M, respectively.

[0063] Example 3: Preparation of fluorescent microsphere-labeled antibodies

[0064] 1. Material preparation

[0065] Azido peptide, Cyclo[RGDfK(Azido)]) also known as Cyclo[Arg-Gly-Asp-D-Phe-Lys(Azido)], cyclopentapeptide, Xi'an Qiyue Biotechnology Co., Ltd. Buffer: PBS at pH 7.4. Catalyst: Catalytic system composed of CuSO4 and sodium ascorbate. Other reagents: Deionized water, ethanol, etc. for washing after reaction.

[0066] Alkyne fluorescent microspheres (CNPCs microspheres) were prepared according to the method in Chapter 2 of “Zhang Peng, Preparation, Characterization and Application of Functionalized Polymer Porous Microspheres, Doctoral Dissertation of Fudan University, 2013”.

[0067] 2. Coupling steps

[0068] Microsphere pretreatment: 5 mg of alkynyl fluorescent microspheres were suspended in 20 mL of PBS buffer and ultrasonicated for 15 min to ensure that the microspheres were evenly dispersed and to avoid agglomeration. A 5 mg / mL azido peptide solution was prepared in PBS.

[0069] 40 mL of azido peptide solution was mixed with 20 mL of alkynyl fluorescent microsphere solution, and copper sulfate with a final concentration of 0.5 mM and 5 mM ascorbic acid were added thereto. The mixture was stirred at room temperature in the dark for 4 h. After the solution was separated by magnetic adsorption, the microspheres were washed several times with PBS buffer to remove unreacted peptides, catalysts and other impurities. Then, deionized water or ethanol was used for the final washing to remove residual salts and other substances to obtain microspheres coupled with cyclopentapeptide. Figure 3As shown, the alkyne absorption peak at 2100~2260 cm −1 basically disappeared, indicating that the azide peptide was successfully conjugated with the alkyne-functionalized fluorescent microspheres.

[0070] 3. Condensed antibody

[0071] Prepare buffers of VP2-VH, VP2-VL, M-VH, and M-VL at 10 mg / mL and a buffer of microspheres conjugated with cyclopentapeptide at 50 mg / mL.

[0072] Add EDC and NHS to the buffers of VP2-VH, VP2-VL, M-VH, and M-VL to a final concentration of 0.2 M and 0.2 M, respectively. After gently stirring at room temperature for 30 min, add 2 volumes of the buffer of microspheres conjugated with cyclopentapeptide to the activated solution of VP2-VH and react at room temperature for 4 h. Then add an equal volume of the activated solution of VP2-VL to the activated solution of VP2-VH and react at room temperature for 4 h. Remove unreacted proteins by magnetic absorption and wash the microspheres with PBS buffer multiple times. Confirm the binding of the peptide to the protein by SDS-PAGE to obtain the VP2 antibody labeled with fluorescent microspheres ( Figure 4 ).

[0073] Add 2 volumes of the buffer of microspheres conjugated with cyclopentapeptide to the activated solution of M-VH and react at room temperature for 4 h. Then add an equal volume of the activated solution of M-VL to the activated solution of M-VH and react at room temperature for 4 h. Remove unreacted proteins by magnetic absorption and wash the microspheres with PBS buffer multiple times. Confirm the binding of the peptide to the protein by SDS-PAGE to obtain the M antibody labeled with fluorescent microspheres ( Figure 4 ).

[0074] 4. Control group

[0075] The magnetic fluorescent microspheres (Zhongke Keyou) were resuspended in pH 6.0 MES buffer, and EDC and 0.2 M sulfo-NHS were added at a final concentration of 0.2 M. The reaction was carried out at room temperature for 30 minutes, and the reaction was gently stirred during the reaction. After the reaction was completed, the microspheres were washed with MES buffer to remove the unreacted activator. A 10 mg / mL buffer of canine parvovirus antibody (MAB8293, R92F6, Merck) or canine coronavirus antibody (A041, Antibio) was prepared, and the buffer was reacted with 2 volumes of 50 mg / mL of the buffer for activating magnetic fluorescent microspheres at room temperature for 4 hours, and then the microspheres were washed with PBS or MES buffer to remove the unbound antibody. The coupled microspheres were resuspended in a blocking solution (such as 1% BSA) and incubated at room temperature for 1 hour. After the blocking was completed, the microspheres were washed with PBS to remove the unbound blocking agent. Resuspend the coupled and blocked magnetic fluorescent microspheres in an appropriate amount of storage solution (such as PBS containing 0.1% BSA) and store at 4°C to avoid repeated freezing and thawing.

[0076] Example 4. Preparation of fluorescent immunochromatographic strips

[0077] The specific preparation steps of the fluorescent immunochromatographic test strip for simultaneously detecting canine parvovirus (CPV) and canine coronavirus (CCV) by fluorescent immunochromatography in this embodiment are as follows:

[0078] 1. Material preparation

[0079] Nitrocellulose membrane, sample pad, conjugate pad, absorbent pad, PVC backing, fluorescent microsphere-labeled VP2 antibody and fluorescent microsphere-labeled M antibody prepared in accordance with the above examples, goat anti-mouse IgG antibody, bovine serum albumin (BSA), Tris-HCl buffer, phosphate buffered saline (PBS), Tween-20 and blocking solution, etc.

[0080] 2. Assembly of test strips

[0081] The buffer containing 1 mg / mL VP2-VH and 1 mg / mL VP2-VL was sprayed on the detection line (T1 line) of the NC membrane using a film dispenser, the buffer containing 1 mg / mL M-VH and M-VL was sprayed on the detection line (T2 line) of the NC membrane, and goat anti-mouse IgG was sprayed on the quality control line (C line). The spraying amount was 0.4 μL / cm. After the spraying was completed, the NC membrane was dried at 37°C for 2 hours. The distance between the T1 and T2 lines was 6 mm, and the distance between the T2 line and the C line was also 6 mm.

[0082] The fluorescent microsphere-labeled VP2 antibody and fluorescent microsphere-labeled M antibody provided by the experimental group were mixed in equal mass and sprayed on the conjugate pad with a dot printer. The spraying concentration was usually 1 mg / mL, and the spraying volume was 0.4 μL / cm. After drying at 37°C for 2 hours, the sample pad and the absorbent pad were respectively pasted on the PVC backing with a nitrocellulose membrane and cut into strips 4 mm wide with a strip cutter to make the test strip, which was then dried and stored for later use. When in use, after the sample was treated to a certain extent, it was dropped onto the sample pad, and a special sample addition hole could be made on the sample pad.

[0083] 3. Test strip of the control group

[0084] Use a dot blotting machine to spray the buffer solution of 1 mg / mL canine parvovirus antibody (MAB8293, R92F6, Merck) on the test line (T1 line) of the NC membrane, and spray the buffer solution of 1 mg / mL canine coronavirus antibody (A041, Antibody Biotech) on the test line (T2 line) of the NC membrane. Spray goat anti-mouse IgG on the quality control line (C line), with a spraying volume of 0.4 μL / cm. After spraying, the NC membrane was dried at 37°C for 2 h. The distance between the T1 and T2 lines was 6 mm, and the distance between the T2 line and the C line was also 6 mm.

[0085] The fluorescent microsphere-labeled VP2 antibody and fluorescent microsphere-labeled M antibody provided by the control group were mixed in equal mass and sprayed on the conjugate pad with a dot printer. The spraying concentration was usually 1 mg / mL, and the spraying volume was 0.4 μL / cm. After drying at 37°C for 2 hours, the sample pad and the absorbent pad were respectively pasted on the PVC backing with a nitrocellulose membrane and cut into strips 4 mm wide with a strip cutter to make the test strip, which was then dried and stored for later use. When in use, after the sample was treated to a certain extent, it was dropped onto the sample pad, and a special sample addition hole could be made on the sample pad.

[0086] Example 5. Detection

[0087] The following are the specific steps for detecting canine parvovirus by fluorescence immunochromatography:

[0088] 1. Sample treatment

[0089] Collect fecal samples of dogs, dilute them with physiological saline, and centrifuge to take the supernatant as the sample to be tested.

[0090] 2. Sample detection

[0091] The test sample is dropped onto the sample pad of the test strip. The sample chromatographs on the test strip and binds to the magnetic fluorescent microspheres on the conjugate pad to form an antigen-antibody complex. The antigen-antibody complex continues to chromatograph to the NC membrane and binds to the sprayed detection antibodies at the test line 1 (T1) and test line 2 (T2) respectively to form a sandwich complex. The control antibody at the control line (C line) is used to verify the effectiveness of the test strip.

[0092] Insert the test strip into the detection window of the fluorescence reader, and the fluorescence intensities of the test line 1 (T1), test line 2 (T2) and control line (C line) will be displayed as numerical values on the display. According to the standard curve already entered in the instrument, the contents of canine parvovirus and canine coronavirus in the sample are calculated to achieve quantitative detection.

[0093] 3. Result interpretation

[0094] If fluorescence or color reactions appear at both the test line 1 (T1) and the control line (C line), it indicates that canine parvovirus exists in the sample. If fluorescence or color reactions appear at both the test line 2 (T2) and the control line (C line), it indicates that canine coronavirus exists in the sample. If fluorescence or color reaction appears only at the control line (C line) and there is no reaction at the test line 1 (T1) and test line 2 (T2), it indicates that neither canine parvovirus nor canine coronavirus exists in the sample.

[0095] 4. Sensitivity test

[0096] Take canine parvovirus VP2 protein or canine coronavirus M protein, dilute it with the sample extraction solution to obtain concentrations of 0, 0.001, 0.01, 0.1, 1, 10 ng / mL. After testing with the test strips prepared in the experimental group and control group in Example 4 respectively, detect the fluorescence intensities of the T1, T2, and C lines with a fluorescence detector. Use the fluorescence intensity ratio of T1 / C or T2 / C as the ordinate and the mass concentrations of different standards as the abscissa to draw the standard curve for the detection of the N-protein of the new coronavirus.

[0097] As a result, as the content of VP2 protein or M protein in the sample increases, the color of the test line T gradually becomes darker. Use logEC50 = logECF - (1 / HillSlope)*log(F / (100 - F)); Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC50 - X)*HillSlope)) for dose-response curve fitting. logEC50 is the logarithm of the half-maximal effective concentration, HillSlope is the Hill slope, and Bottom and Top are the bottom and top of the curve respectively. As Figure 5It can be seen that the curves of the test strips in the experimental group for detecting VP2 protein and M protein both have very good fitting effects, and the correlation coefficients are 0.9974 and 0.9961 respectively. The curves of the test strips in the control group for detecting VP2 protein and M protein both have very good fitting effects, and the correlation coefficients are 0.9989 and 0.9989 respectively. However, the T / C average value of the blank sample without antigen is 0.002. When the control group's test strips detect samples of 10 -5 ng / mL and 10 -4 ng / mL, there is no obvious difference in their T / C values from the blank sample, indicating that the test strips provided by the control group cannot detect 10 -5 ng / mL and 10 -4 ng / mL of VP2 protein or 10 -5 ng / mL and 10 -4 ng / mL of M protein, and its sensitivity is lower than that of the experimental group.

[0098] 5. Specificity test

[0099] The recombinant porcine parvovirus VP2 protein (PhyCell®), novel coronavirus N protein (Dongguan Bio-Protech Co., Ltd.), canine parvovirus VP2 protein, and canine coronavirus M protein were respectively prepared into sample solutions of 10 ng / mL. After testing with the test strips prepared by the experimental group and the control group in Example 4 respectively, the T1, T2, and C lines were shown by a fluorescence detector. As Figure 6 shown, the test strips provided by the experimental group did not show color for the recombinant porcine parvovirus VP2 protein and the novel coronavirus N protein, but showed obvious fluorescence on the T1 line and T2 line for the canine parvovirus VP2 protein and the canine coronavirus M protein respectively, indicating that the experimental group has obvious specificity for the canine parvovirus VP2 protein and the canine coronavirus M protein respectively and can exclude the interference of the recombinant porcine parvovirus VP2 protein and the novel coronavirus N protein. In addition, although the test strips of the control group can show obvious fluorescence for the canine parvovirus VP2 protein and the canine coronavirus M protein, the T2 line for detecting the canine parvovirus VP2 protein is also shown, indicating that its own specificity for the canine parvovirus VP2 protein and the canine coronavirus M protein is insufficient and it cannot effectively exclude the interference of the two. In addition, the test strips of the control group also showed fluorescence for the recombinant porcine parvovirus VP2 protein and could not exclude its interference.

[0100] 6. Actual sample testing

[0101] The test strips provided by the experimental group and the control group of the present invention were used to simultaneously collect canine fecal separation samples (for detecting canine parvovirus) and throat swab samples (for detecting canine coronavirus) for clinical comparison tests. In addition, a canine parvovirus (CPV) nucleic acid detection kit (PCR-fluorescent probe method, product number YB-11-1200, Yubo Biology) was used to detect these canine separation samples, and a canine respiratory coronavirus RT-PCR kit (product number XY-P0055, XYBIO) was used to detect these throat swab samples.

[0102] For canine parvovirus, the sensitivity of the experimental group was 92.35%, 95%CI: 89.21%~98.92%; the specificity was 95.35%, 95%CI: 90.26%~98.16%. The sensitivity of the control group was 79.26%, 95%CI: 61.82%~85.36%; the specificity was 68.49%, 95%CI: 58.39%~79.26%. The sensitivity of the PCR-fluorescent probe method was 90.83%, 95%CI: 84.76%~93.29%; the specificity was 92.79%, 95%CI: 89.44%~96.09%.

[0103] For canine coronavirus, the sensitivity of the experimental group was 90.95%, 95%CI: 86.82%~94.33%; the specificity was 96.18%, 95%CI: 92.48%~99.08%. The sensitivity of the control group was 82.36%, 95%CI: 75.93%~89.08%; the specificity was 72.64%, 95%CI: 63.09%~81.82%. The sensitivity of the PCR-fluorescent probe method was 86.92%, 95%CI: 81.83%~92.39%; the specificity was 91.48%, 95%CI: 87.98%~95.42%.

[0104] The test results show that the test strips and detection kits for canine parvovirus / canine coronavirus developed by the present invention using fluorescence immunochromatography have good sensitivity and specificity, and a high coincidence rate with clinical diagnosis, and can be used as an effective screening and diagnostic technical means for canine parvovirus / canine coronavirus infection.

[0105] As described above, it is only the preferred specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by this application should be covered within the protection scope of this application.

Claims

1. A fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit, characterized in that: Including fluorescent microsphere labeled VP2 antibody, fluorescent microsphere labeled M antibody, goat anti-mouse IgG antibody, nitrocellulose membrane, sample pad, conjugate pad, absorbent pad, PVC backing; The fluorescent microsphere-labeled VP2 antibody is an antibody obtained by coupling the VP2-VH shown in SEQ ID NO:1 and the VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys, and the fluorescent microsphere-labeled M antibody is an antibody obtained by coupling the M-VH shown in SEQ ID NO:3 and the M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys.

2. A method for assembling a fluorescent immunochromatography canine parvovirus / canine coronavirus test strip, characterized in that: include: The buffer containing VP2-VH and VP2-VL was sprayed on the T1 detection line of the NC membrane, the buffer containing M-VH and M-VL was sprayed on the T2 detection line of the NC membrane, and the goat anti-mouse IgG was sprayed on the quality control line. After the spraying was completed, the NC membrane was dried at 37°C for 2h. The distance between the T1 and T2 lines was 6 mm, and the distance between the T2 line and the C line was also 6mm; VP2 antibody labeled with fluorescent microspheres and M antibody labeled with fluorescent microspheres were mixed in equal amounts and sprayed on the conjugate pad using a film dispenser. After spraying, the NC membrane was dried at 37°C for 2 h. The sample pad and absorbent pad were respectively pasted on the PVC backing with nitrocellulose membrane and cut into 4 mm wide strips with a strip cutter to make test strips; Among them, the fluorescent microsphere-labeled VP2 antibody is an antibody obtained by coupling the VP2-VH shown in SEQ ID NO:1 and the VP2-VL shown in SEQ ID NO:2 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys, and the fluorescent microsphere-labeled M antibody is an antibody obtained by coupling the M-VH shown in SEQ ID NO:3 and the M-VL shown in SEQ ID NO:4 to magnetic fluorescent microspheres carrying Arg-Gly-Asp-D-Phe-Lys.

3. The fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit according to claim 1, characterized in that: The VP2-VH shown in SEQ ID NO:1 is prepared by cloning the target sequence shown in SEQ ID NO:5 into pET-28a(+) to obtain an expression plasmid pET-28a-VP2-VH, transforming pET-28a-VP2-VH into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VH from the lysate; The VP2-VL shown in SEQ ID NO:2 is prepared by cloning the target sequence shown in SEQ ID NO:6 into pET-28a(+) to obtain the expression plasmid pET-28a-VP2-VL, transforming pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting the bacteria from the culture medium, lysing the bacteria, and purifying and collecting the VP2-VL from the lysate.

4. The fluorescent immunochromatography canine parvovirus / canine coronavirus detection kit according to claim 1, characterized in that: The VP2-VH shown in SEQ ID NO:3 is prepared by cloning the target sequence shown in SEQ ID NO:7 into pET-28a(+) to obtain an expression plasmid pET-28a-VP2-VH, transforming pET-28a-VP2-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting bacterial cells from the culture solution, lysing the bacterial cells, and purifying and collecting the VP2-VH from the lysate; The M-VL shown in SEQ ID NO:4 is obtained by cloning the target sequence shown in SEQ ID NO:8 into pET-28a(+) to obtain an expression plasmid pET-28a-M-VL, transforming pET-28a-M-VL into Escherichia coli, screening positive clones, culturing and inducing expression of the positive clones, collecting bacteria from the culture medium, lysing the bacteria, and purifying and collecting the M-VL from the lysate.

5. The assembly method according to claim 2, characterized in that: The fluorescent microsphere-labeled VP2 antibody is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azido peptide to obtain cyclopentapeptide-coupled microspheres; then reacting the VP2-VH activation solution with the cyclopentapeptide-coupled microspheres, and then adding the VP2-VL activation solution to react; The fluorescent microsphere-labeled M antibody is obtained by reacting alkynyl fluorescent microspheres with Arg-Gly-Asp-D-Phe-Lys azido peptide to obtain microspheres coupled with cyclopentapeptide; then reacting the M-VH activation solution with the microspheres coupled with cyclopentapeptide, and then adding the M-VL activation solution to react.

6. Use of antibodies against canine parvovirus and / or antibodies against canine coronavirus in the preparation of a canine parvovirus / canine coronavirus detection kit by fluorescent immunochromatography, wherein the antibodies against canine parvovirus include VP2-VH as shown in SEQ ID NO: 1 and VP2-VL as shown in SEQ ID NO: 2, and the antibodies against canine coronavirus include M-VH as shown in SEQ ID NO: 3 and M-VL as shown in SEQ ID NO: 4.

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